Non-genotoxic Liver Carcinogen Research Articles

Gene expression and cytosine DNA methylation alterations in induced pluripotent stem-cell-derived human hepatocytes treated with low doses of chemical carcinogens.

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk emphasizes the need to develop reliable time- and cost-effective approaches for carcinogen detection. To address this issue, we have investigated the utility of human hepatocytes for the in vitro identification of genotoxic and non-genotoxic carcinogens. Induced pluripotent stem-cell (iPSC)-derived human hepatocytes were treated with the genotoxic carcinogens aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P), the non-genotoxic liver carcinogen methapyrilene, and the non-carcinogens aflatoxin B2 (AFB2) and benzo[e]pyrene (B[e]P) at non-cytotoxic concentrations for 7days, and transcriptomic and DNA methylation profiles were examined. 1569, 1693, and 2061 differentially expressed genes (DEGs) were detected in cells treated with AFB1, B[a]P, and methapyrilene, respectively, whereas no DEGs were found in cells treated with AFB2 or B[e]P. In contrast to the profound cellular transcriptomic responses, exposure of iPSC-derived hepatocytes to the test chemicals resulted in minor random alterations in global DNA methylome, most of which were not associated with changes in gene expression. Overall, our results demonstrate that the major non-genotoxic effect of exposure to carcinogens, regardless of their mode of action, is a profound global transcriptomic response rather than global DNA methylome alterations, indicating the significance of transcriptomic alterations as an informative endpoint in short-term in vitro carcinogen testing.

Effect of aflatoxin B1, benzo[a]pyrene, and methapyrilene on transcriptomic and epigenetic alterations in human liver HepaRG cells

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk highlights the need for developing reliable time- and cost-effective approaches for carcinogen detection and identification. To address this issue, we investigated the utility of high-throughput microarray gene expression and next-generation genome-wide DNA methylation sequencing for the in vitro identification of genotoxic and non-genotoxic carcinogens. Terminally differentiated and metabolically competent human liver HepaRG cells were treated at minimally cytotoxic concentrations of (i) the genotoxic human liver carcinogen aflatoxin B1 (AFB1) and its structural non-carcinogenic analog aflatoxin B2 (AFB2); (ii) the genotoxic human lung carcinogen benzo[a]pyrene (B[a]P) and its non-carcinogenic isomer benzo[e]pyrene (B[e]P); and (iii) the non-genotoxic liver carcinogen methapyrilene for 72 h and transcriptomic and DNA methylation profiles were examined. Treatment of HepaRG cells with the liver carcinogens AFB1 and methapyrilene generated distinct gene-expression profiles, whereas B[a]P had only a slight effect on gene expression. In contrast to transcriptomic alterations, treatment of HepaRG cells with the carcinogenic and non-carcinogenic chemicals resulted in profound changes in the DNA methylation footprint; however, the correlation between gene-specific DNA methylation and gene expression changes was minimal. Among the carcinogen-altered genes, transferrin (TF) emerged as sensitive marker for an initial screening of chemicals for their potential liver carcinogenicity. Potential liver carcinogens (i.e., chemicals causing altered TF gene expression) could then be subjected to gene-expression analyses to differentiate genotoxic from non-genotoxic liver carcinogens. This approach may substantially enhance the identification and assessment of potential liver carcinogens.

Oxidative Stress Mechanisms Do Not Discriminate between Genotoxic and Nongenotoxic Liver Carcinogens

It is widely accepted that in chemical carcinogenesis different modes-of-action exist, e.g., genotoxic (GTX) versus nongenotoxic (NGTX) carcinogenesis. In this context, it has been suggested that oxidative stress response pathways are typical for NGTX carcinogenesis. To evaluate this, we examined oxidative stress-related changes in gene expression, cell cycle distribution, and (oxidative) DNA damage in human hepatoma cells (HepG2) exposed to GTX-, NGTX-, and noncarcinogens, at multiple time points (4-8-24-48-72 h). Two GTX (azathriopine (AZA) and furan) and two NGTX (tetradecanoyl-phorbol-acetate, (TPA) and tetrachloroethylene (TCE)) carcinogens as well as two noncarcinogens (diazinon (DZN, d-mannitol (Dman)) were selected, while per class one compound was deemed to induce oxidative stress and the other not. Oxidative stressors AZA, TPA, and DZN induced a 10-fold higher number of gene expression changes over time compared to those of furan, TCE, or Dman treatment. Genes commonly expressed among AZA, TPA, and DZN were specifically involved in oxidative stress, DNA damage, and immune responses. However, differences in gene expression between GTX and NGTX carcinogens did not correlate to oxidative stress or DNA damage but could instead be assigned to compound-specific characteristics. This conclusion was underlined by results from functional readouts on ROS formation and (oxidative) DNA damage. Therefore, oxidative stress may represent the underlying cause for increased risk of liver toxicity and even carcinogenesis; however, it does not discriminate between GTX and NGTX carcinogens.

Gene array screening for identification of drugs with low levels of adverse side effects

One of the success stories of modern toxicology is the identiWcation of gene expression patterns that are associated with speciWc toxic mechanisms (Ellinger-Ziegelbauer et al. 2008; Sano et al. 2009; Dika Nguea et al. 2008). A wellknown example is the classiWcation algorithm diVerentiating between genotoxic and non-genotoxic liver carcinogens as opposed to non-carcinogens (Ellinger-Ziegelbauer et al. 2004, 2005, 2008). However, many more compounds or classes of compounds have been shown to be associated with speciWc gene expression patterns (Tsukue et al. 2009; Schug et al. 2008; Glahn et al. 2008; Hewitt et al. 2007). In this issue of the Archives of Toxicology, JeVrey F. Waring et al. from Abbott Laboratories present another example of high practical relevance (Waring et al. 2010). In recent years, protease inhibitors have contributed to the reduction of the morbidity and mortality caused by AIDS. These protease inhibitors block cleavage of the gag and gag-pol protein precursors that are required for HIV virus maturation. However, these drugs are associated with adverse side eVects. For example, results from clinical studies show an increase in cholesterol and triglyceride levels. Therefore, Waring and colleagues have administered protease inhibitors, which either cause lipid elevations or are relatively lipid neutral. Analyzing gene expression patterns in the liver of exposed rats, they identiWed increased expression of genes encoding proteasomal subunits speciWcally in the rats exposed to protease inhibitors known to cause lipid elevations. Based on this observation, the authors speciWcally screened for protease inhibitors that do not induce the proteasomal genes. Their eVorts led to the identiWcation of a novel protease inhibitor, which did not upregulate proteasomal marker genes and, therefore, may represent a “lipid neutral” compound. It will be very interesting to see whether this prediction can be conWrmed in clinical studies.

Global liver proteomics of rats exposed for 5 days to phenobarbital identifies changes associated with cancer and with CYP metabolism.

A global proteomics approach was applied to model the hepatic response elicited by the toxicologically well-characterized xenobiotic phenobarbital (PB), a prototypical inducer of hepatic xenobiotic metabolizing enzymes and a well-known nongenotoxic liver carcinogen in rats. Differential detergent fractionation two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry and systems biology modeling were used to identify alterations in toxicologically relevant hepatic molecular functions and biological processes in the livers of rats following a 5-day exposure to PB at 80 mg/kg/day or a vehicle control. Of the 3342 proteins identified, expression of 121 (3.6% of the total proteins) was significantly increased and 127 (3.8%) significantly decreased in the PB group compared to controls. The greatest increase was seen for cytochrome P450 (CYP) 2B2 (167-fold). All proteins with statistically significant differences from control were then analyzed using both Gene Ontology (GO) and Ingenuity Pathways Analysis (IPA, 5.0 IPA-Tox) for cellular location, function, network connectivity, and possible disease processes, especially as they relate to CYP-mediated metabolism and nongenotoxic carcinogenesis mechanisms. The GO results suggested that PB's mechanism of nongenotoxic carcinogenesis involves both increased xenobiotic metabolism, especially induction of the 2B subfamily of CYP enzymes, and increased cell cycle activity. Apoptosis, however, also increased, perhaps, as an attempt to counter the rising cancer threat. Of the IPA-mapped proteins, 41 have functions which are procarcinogenic and 14 anticarcinogenic according to the hypothesized nongenotoxic mechanism of imbalance between apoptosis and cellular proliferation. Twenty-two additional IPA nodes can be classified as procarcinogenic by the competing theory of increased metabolism resulting in the formation of reactive oxygen species. Since the systems biology modeling corresponded well to PB effects previously elucidated via more traditional methods, the global proteomic approach is proposed as a new screening methodology that can be incorporated into future toxicological studies.

Gene Expression Dose-Response of Liver with a Genotoxic and Nongenotoxic Carcinogen

Tumorigenic mechanisms due to chemical exposure are broadly classified as either genotoxic or nongenotoxic. Genotoxic mechanisms are generally well defined; however nongenotoxic modes of tumorgenesis are less straightforward. This study was undertaken to help elucidate dose-response changes in gene expression (transcriptome) in the liver of rats in response to administration of known genotoxic or nongenotoxic liver carcinogens. Male Big Blue Fischer 344 rats were treated for 28-days with 0, 0.1, 0.3, 1.0, or 3.0 mg/kg/day of the genotoxin 2-acetylaminofluorene (AAF) or 0, 10, 30, 60, or 100 mg/kg/day of the nongenotoxin phenobarbital (PB). Transcriptome analysis was performed using the relatively focused Clontech Rat Toxicology II microarray (465 genes) and hybridized with 32P-labeled cDNA target. The analysis indicated that after 28 days of treatment, AAF altered the expression of 14 genes (9 up- and 5 down-regulated) and PB altered the expression of 18 genes (10 up- and 8 down-regulated). Of the limited genes whose expression was altered by AAF and PB, four were altered in common, two up-regulated, and two down-regulated. Several of the genes that show modulation of transcriptional activity following AAF and PB treatment display an atypical dose-response relationship such that the expression at the higher doses tended to be similar to that of control. This high-dose effect could potentially be caused by adaptation, toxicity, or tissue remodeling. These results suggest that the transcriptional response of the cells to higher doses of a toxic agent is likely to be different from that of a low-dose exposure.

The Slow Induction of Resistant Hepatocytes during Initiation of Hepatocarcinogenesis by the Nongenotoxic Carcinogen Clofibrate

This study was designed to explore whether a well-known nongenotoxic liver carcinogen, clofibrate, would induce rare resistant hepatocytes similar to those seen during initiation of hepatocarcinogenesis with many genotoxic carcinogens. Male young adult F344 rats were exposed to a control diet containing 0.5% (w/w) clofibrate for 3, 6, or 10 months. After 1 month on a diet free of clofibrate, the animals were assayed for resistant hepatocytes by a standardized selection procedure using 2-acetylaminofluorene as the inhibitor and partial hepatectomy as a strong stimulus for cell proliferation. No resistant hepatocytes were found in the animals exposed to clofibrate for 3 months or in any of a series of control animals. However, animals on the clofibrate for 6 and 10 months contained resistant hepatocytes that were clonally expanded to produce hepatocyte nodules. These nodules were indistinguishable on gross and microscopic examination from hepatocyte nodules seen in animals in which nodules are induced with one of many different genotoxic carcinogens. Also, like those nodules, the nodules seen in the animals exposed to clofibrate stained positively for glutathione S-transferase 1-1 and γ-glutamyl transpeptidase and negatively for ATPase. The evidence from this study indicates that the nongenotoxic carcinogen, clofibrate, induces early cellular changes in the liver that are very similar to those induced by many different genotoxic carcinogens. These changes are manifest as a resistance phenotype in a few scattered hepatocytes that now can be clonally expanded selectively to form hepatocyte nodules. However, the resistant hepatocytes are induced by clofibrate much more slowly. Whether this basic similarity pertains to the later steps in the hepatocarcinogenic process remains to be studied.

Cytokines in non-genotoxic hepatocarcinogenesis.

Many toxicants can cause liver injury. Some, such as diethylnitrosamine, are genotoxic and act principally by damaging DNA (1). A second more diverse group cause liver injury but are non-genotoxic (2). Despite differences in the primary target, genotoxic and non-genotoxic hepatotoxicants frequently cause tumours in the liver of experimental rats and mice (1,2). This review is concerned with evaluating the hypothesis that cytokines may play a role in mediating the biological effects of liver non-genotoxic carcinogens. Non-genotoxic liver toxicants constitute a diverse group of chemicals. Some, such as carbon tetrachloride (3), cause acute liver injury followed by regenerative hyperplasia, whereas others cause proliferation without identifiable tissue damage. The latter group includes barbiturate drugs such as phenobarbitone (PB) (4), dioxins such as 2,3,7,8-tetrachlorodibenzo-pdioxin (5,6) and the largest group, the peroxisome proliferators (PPs) (7). The induction of cell proliferation by toxicants that cause liver injury probably underpins their common ability to cause hepatocellular adenomas and carcinomas after prolonged exposure (8). During chemical-induced carcinogenesis, the tightly controlled balance that exists normally in tissues between cell growth and cell death is disrupted (9–11). Rodent hepatocarcinogenesis in response to PPs is preceded by liver enlargement due to hepatocyte hypertrophy and induction of cell proliferation coupled with suppression of hepatocyte apoptosis (7,12,13). The ability of barbiturates such as PB to stimulate hepatocyte DNA synthesis in vivo has also been demonstrated (14). The capacity of non-genotoxic liver carcinogens to perturb both sides of the growth equation between cell gain and cell loss can be modelled in vitro using hepatocytes prepared from rats or mice. In such models, PPs such as nafenopin can suppress spontaneous apoptosis (15–17) and also that induced by diverse stimuli, including transforming growth factor-β1. Interleukin (IL)-1 can act alongside TNFα to play a role in inflammation (18) and is implicated in the regulation of hepatic lipid metabolism (19), ligation of Fas or DNA damage (20). Thus, non-genotoxic carcinogens may

Non-genotoxic hepatocarcinogens stimulate DNA synthesis and their withdrawal induces apoptosis, but in different hepatocyte populations.

Non-genotoxic hepatocarcinogenesis may involve suppression of the hepatocyte apoptosis that would normally remove damaged or initiated cells. These protected hepatocytes could then remain as preferential targets for promotion by this class of compounds. Here we demonstrate clearly that the non-genotoxic liver carcinogens and hepatomitogens cyproterone acetate (CPA) and nafenopin, a peroxisome proliferator, both suppress the basal level of rat liver apoptosis in vivo. After 10 days of dosing with CPA (120 mg/kg/day) or nafenopin (25 mg/kg/day) there were 0.005 +/- 0.010 and 0.002 +/- 0.021 apoptotic bodies/100 hepatocytes respectively, compared with 0.031 +/- 0.008 per 100 in controls. Concomitant with this suppression of apoptosis, bromodeoxyuridine (BrdU) labelling indices and mitotic figures rose, confirming a perturbation of both sides of the growth equation between cell death and replication. Withdrawal of CPA or nafenopin resulted in a 100- to 200-fold elevation in apoptosis. This was inhibited by the re-administration of either compound. To investigate if cells protected from apoptosis by non-genotoxic carcinogens are targets for replication, we examined the replicative history of the apoptotic bodies generated upon withdrawal of CPA or nafenopin. Rats were administered BrdU during the hyperplastic phase of compound administration (0-10 days). Livers were examined 5 days after compound withdrawal. With both CPA and nafenopin, apoptotic bodies and S phase were predominantly in the periportal region. However, despite this zonal co-localization, very few (< 10%) of the apoptotic bodies were labelled with BrdU. Overall, our data provide in vivo evidence to support the hypothesis that non-genotoxic hepatocarcinogens such as CPA and the peroxisome proliferators suppress apoptosis. Surprisingly, the majority of the hepatocytes generated during compound-induced hyperplasia were protected from apoptosis during liver regression. These data contribute to our understanding of clonal selection and promotion during non-genotoxic hepatocarcinogenesis.

The carcinogenic effect of methapyrilene combined with nitrosodiethylamine given to rats in low doses.

The carcinogenic effects of combinations of methapyrilene hydrochloride (MP), nitrosodiethylamine (NDEA), and phenobarbital (PB) or partial hepatectomy (PH) were examined following sequential treatment of rats. MP is a generally non-genotoxic liver carcinogen of moderate potency, NDEA is a genotoxic liver carcinogen, PB is primarily a liver tumor promoter and PH induces cell proliferation. The dose of each carcinogen was chosen to be below that causing significant liver tumor incidence when given singly. There were 12 protocols involving groups of 28 female rats each. Short treatments with NDEA and MP were followed by 60 weeks of PB promotion or by partial hepatectomy. Each treatment was given separately or in double combination as controls. Several animals of each group were killed at intervals during the experiment for examination of toxic effects and the presence of altered hepatic foci. In only 3 of 12 groups was there a significant incidence of rats with liver neoplasms: the two groups given three treatments: NDEA, MP and PB (86% tumors) or NDEA, MP and PH (33%), and the group receiving NDEA and MP without promotion (46%). The results clearly indicated a co-carcinogenic effect between NDEA and MP. Continuous PB potentiated tumor development, while PH did not. There was no evidence of liver toxicity from any of the treatments, but clear cell foci observed in three groups at weeks 13 and 33 correlated with the later development of liver neoplasms.

Ultrastructural changes in the liver of animals treated with methapyrilene and some analogs

Methapyrilene is a liver carcinogen in rats and induces mitochondrial proliferation in rat liver, but appears to have no genotoxic or other effects that can be related to its carcinogenic action. A number of structural analogs of methapyrilene were administered to rats and the livers were examined for ultrastructural changes similar to those induced by methapyrilene. Only one analog, methafurylene, produced a small but not significant increase in mitochondria. Pyrilamine, which has not induced liver tumors in rats, produced no detectable changes visible in the electron microscope. The nongenotoxic liver carcinogen nitrosodiethanolamine did not induce mitochondrial proliferation in rat liver. In Syrian hamsters and guinea pigs methapyrilene did not induce ultrastructural changes in the liver comparable with those seen in rats and this compound has not induced tumors in these species. The ultrastructural changes induced in rat liver by methapyrilene seem, therefore, to be unique to this compound.